1. Field of the Invention
The invention relates to fluorescent dyes that are useful in various assays and, more particularly, to fluorescent polymeric dyes, wherein fluorescence is enhanced by at least partially hosting fluorophoric moieties with moieties providing a hydrophobic and conformationally restrictive microenvironment.
2. Discussion of the Art
A variety of assay techniques are employed in quantitative and qualitative analysis of chemical and biochemical mixtures. One assay technique, referred to as "fluorescence", is useful in many biochemical studies. This assay technique utilizes a fluorescent chemical to label certain molecules to distinguish those molecules from unlabeled, but similar, molecules. A chemical is considered to be fluorescent if it absorbs light at a given wavelength (the "excitation" wavelength) and emits light at a longer wavelength (the "emission" wavelength). The fluorescent chemicals used in this type of assay are often referred to as fluorescent dyes.
There are numerous optical techniques for detecting fluorescent dyes employed in fluorescence assays. One such technique is flow cytometry. Flow cytometry is employed in fluorescence assays to identify particular molecules or cells and to separate or distinguish those molecules or cells from a mixture. In a typical flow cytometry procedure, a fluorescent dye is linked to an antibody. The antibody is specific to an antigen of a particular molecule or a cell-surface molecule of a particular cell desired to be detected. The linking of the antibody and fluorescent dye is referred to as "conjugation", and the linked antibody-fluorescent dye complex is referred to as a "conjugate".
After an appropriate antibody and an appropriate dye are linked to form a conjugate, the conjugate is added to a mixture suspected of containing the antigen or cell-surface molecule sought to be detected. When the conjugate is added to the mixture and appropriate conditions are maintained, the antibody of the conjugate binds with the antigen or cell-surface molecule. The entire mixture in which the antigen or cell-surface molecule is contained and to which the conjugate was added is then subjected to a laser beam of the excitation wavelength for the particular fluorescent dye. The laser beam of this wavelength causes the molecules or cells that contain the bound antibody-fluorescent dye conjugate to fluoresce. A flow cytometer may detect and measure the amount of laser light scattered by the bound molecule or cell, and by that measurement, the quantity, quality, and other determinations relating to the detected antigen or cell-surface molecule may be made.
It has typically been necessary to take steps to increase the intensity of the fluorescent dyes for better detection. Several means have been employed to increase the intensity. However, there are significant limitations that reduce the effectiveness of those means.
One means for increasing the intensity of fluorescent dyes at a given wavelength has been the mechanism of fluorescence energy transfer, whereby a transfer of energy from an excited state is made from a donor molecule to an acceptor molecule. The transfer is usually accomplished by positioning one fluorophore close to another fluorophore. As used herein, the expression "fluorophore" means a carrier of fluorescence.
The first of the closely-positioned fluorophores may be excited by light of a given wavelength. Then, instead of emitting light of a longer wavelength, the excited fluorophore transfers energy to the second fluorophore. That transferred energy excites the second fluorophore, which then emits light of an even longer wavelength than would have been emitted by the first fluorophore. An example of such an energy transfer arrangement involves phycobiliprotein-cyanine dye conjugates. Subjecting these conjugates to an about 488 nm laser light excites the phycobiliprotein. The phycobiliprotein will then, without itself irradiating, transfer energy to the cyanine fluorophore at the excitation wavelength of the cyanine, which is coincident with the emission wavelength of the phycobiliprotein, about 580 nm. Consequently, the cyanine fluorophore is thereby excited and subsequently emits light of its emission wavelength of about 680 nm. This type of energy transfer system in often referred to as a "tandem energy transfer system."
Energy transfer is not a very simple means for increasing fluorescence for a number of reasons. Two fundamental requirements in energy transfer are an appropriate relative spatial distance relationship of the donor and acceptor molecules and an appropriate relative angular relationship of the absorption and emission dipoles of the two molecules. Obtaining and maintaining these fundamental relationships is extremely difficult, if not impossible, in many circumstances. Additionally, there are many other requirements, including overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor, stability of the fluorophores, change in fluorescent characteristics upon conjugation, quantum efficiency of the transfer, non-specific binding of the fluorophores to other compounds, and others. Eliminating the need for meeting these requirements would be an improvement in the art.
Another means for increasing fluorescent intensity of fluorescent dyes is to attach a multiplicity of fluorophores to a polymer and attach the polymer to an antibody. In this arrangement, each of the fluorophores attached to the polymer may be excited by a laser light and emit light at its emission wavelength. However, the use of polymers for this purpose has generally not been effective. The primary problem encountered with polymers is that when a multiplicity of fluorophores are randomly placed on a single polymer, signal quenching among the fluorophores results. Further, even if the polymer/antibody conjugate emits a greater cumulative quantity of light due to the multiplicity of fluorophores, the emission wavelength is only that applicable to the particular fluorophores. Fluorophores of the prior art have had a limited range of wavelength variation between excitation wavelength and emission wavelength. It would be desirable to provide both a polymer to which may be attached a multiplicity of fluorophores without quenching and a fluorescent dye that emits light of a wavelength much greater than the excitation wavelength. Such a polymeric arrangement and wavelength range would enable more accurate detection.
Yet another means for increasing the intensity of fluorescent dyes involves the use of cyclodextrins. Cyclodextrins are well known water soluble cyclic oligosaccharides having a hydrophobic central cavity and a hydrophilic peripheral region. Generally, the shape of a cyclodextrin molecule is substantially cylindrical, with one end of the cylinder having a larger opening than the other. The smaller opening is known as the primary rim, and the larger opening is known as the secondary rim. A cavity into which small molecules can enter through the larger secondary rim is present between the two openings of the cyclodextrin molecule and, in aqueous systems, this cavity of a cyclodextrin molecule provides a hydrophobic microenvironment for the complexing of hydrophobic molecules of low molecular weight. The cyclodextrin molecule acts as a host for the hydrophobic molecule of low molecular weight, i.e., the guest.
Efforts to generate polymeric cyclodextrins have been made in an attempt to increase the fluorescence associated with fluorophores. Theoretically, the complexing properties of a single cyclodextrin molecule can be magnified by having several cyclodextrin molecules in close proximity to each other, for the reason that having several cyclodextrin molecules in close proximity to each other increases the probability that a guest molecule will enter the cavity of a cyclodextrin molecule. According to the theory, if a polymeric cyclodextrin molecule were created, it would be capable of hosting a plurality of guest molecules. Further, if the guest molecules were signal-generating groups, there would be several fluorophores in close proximity to each other, and the fluorescence associated with the polymer would be greater than that of a single fluorophore. Hence, according to the theory, if a conjugate were made with a polymeric cyclodextrin containing a plurality of fluorophores, fluorescence of the polymer would be greater than that of a conjugate comprising a single fluorophore.
Several polymeric cyclodextrins have been manufactured to validate the above-described theory. However, those polymers suffer from problems that severely limit their effectiveness. The polymeric cyclodextrins are synthesized by using cyclodextrin monomers that have been modified to contain several reactive groups on the cyclodextrin monomer's primary and secondary rims, thereby allowing these monomers to react via their primary and secondary rims, and react multiple times via their multiple reactive groups. When a cyclodextrin molecule is bound by its secondary rim, the larger opening to the hydrophobic cavity is hindered. As a result, it is difficult for a guest molecule to enter the cavity of the cyclodextrin molecule, and the cyclodextrin's utility as a host is compromised. Further, polymers derived from cyclodextrin monomers having multiple reactive groups results in a high degree of cross-linking. When cross-linking occurs, not only are the cyclodextrin molecules bound by the secondary rims, causing the problems mentioned previously, but a matrix of cyclodextrins forms. Consequently, the number of cyclodextrin monomers polymerized is limited and many of the cyclodextrin monomers polymerized become buried within the matrix. Although many cyclodextrin molecules are in close proximity, very few of them have accessible secondary openings and very few guest/host complexes are able to form.
Another means for increasing intensity of fluorescent dyes is judicious selection of a suitable dye, among the many fluorescent dyes from which to choose. It has been common practice to employ naturally-occurring substances as fluorescent dyes for fluorescence testing. The more common naturally-occurring dyes include the phycobiliproteins, such as phycoerythrin, and others. As previously mentioned in connection with the discussion of energy transfer, phycobiliproteins are still being used in some tandem energy transfer systems. However, phycobiliproteins present certain problems in their use. A particular problem with phycobiliproteins is their instability. Exposure to light and other environmental effects can cause photo-bleaching, thereby adversely affecting fluorescence assays.
In recent years, a number of synthetic fluorescent dyes have been manufactured and employed for fluorescence assays. A well-known class of fluorescent dyes are the cyanine dyes. These dyes are polymethine dyes containing the --N.paren open-st.C.dbd.C--C.dbd.).sub.n N--moiety.
Cyanine fluorescent dyes also present problems when employed in fluorescence biological testing procedures, such as flow cytometry. For example, many of these dyes are expensive to use and difficult to manufacture. Further, many of the cyanine dyes do not have a sufficiently large interval, i.e., Stokes' shift, between their excitation wavelength and their emission wavelength to be effective for fluorescence detection methods without utilizing energy transfer involving another fluorophore. Those dyes that do have a sufficiently large interval between excitation wavelength and emission wavelength are often sensitive to the environment.
Another class of fluorescent dyes that has been considered for use in biological testing procedures includes the aminostyryl pyridinium dyes. Because of environmental sensitivity, these dyes have been considered unsuitable for fluorescence labeling applications, such as flow cytometry. The environmental sensitivity of aminostyryl pyridinium dyes is well studied and described by Anthony C. Stevens et al., "Synthesis of Protein-Reactive (Aminostyryl)pyridinium Dyes", Bioconjugate Chem. 1993, 4, 19-24.
It would be desirable to develop a fluorescent dye whereby the need for energy transfer is eliminated and problems associated with environmental sensitivity are overcome.